1. Field of the Invention
The present invention relates to the interaction between hydrophobic compounds and mammalian cells. It includes the areas of in vitro testing of the effects of hydrophobic compounds on cells for biomedical research purposes, including cancer research and genotoxicity assessment, and for the production and application of new pharmaceutical compositions containing hydrophobic substances for administration in liquid or powder form.
2. Description of Related Art
The study of aliphatic hydrocarbons (alkanes) has become increasingly important in biomedical research (1-3). The alkane components of mineral paraffin oils are implicated in a number of human pathologic conditions such as mineral oil pneumonia, folliculitis of the skin, oleogranuloma (paraffinoma), and systemic focal lipidosis (4). Human exposure to mineral paraffin oils comes from workplace contaminants (5), environmental pollutants (6), food (7), and medications (8). Particular interest in the biological actions of alkanes derives from the fact that in genetically susceptible inbred strains of mice (BALB/c and NZB), intraperitoneal injection of mineral oils such as the isoalkane pristane (2,6,10,14-tetramethylpentadecane) induces a malignant B cell lymphoma (plasmacytoma, 9). Over the years, pristane-induced plasmacytomagenesis has become the most intensively studied prototype for plasma cell tumorigenesis (reviewed in 10 and 11).
Pristane and other lipophilic alkanes are practically immiscible with aqueous media (12) required for cell growth and viability. However, when pristane is injected and then withdrawn from the peritoneal cavity of a mouse it appears as an emulsion even after only a few days. Emulsification of pristane in vivo is probably facilitated by amphiphilic molecules (e.g., fatty acids, fatty alcohols, phospholipids, and proteins) that are present in abundance in the inflammatory environment that results from injection of the oil. In a conventional plasmacytoma induction protocol, 1.5 ml of pure pristane (.apprxeq.4.4 mmol) are injected. The oil is gradually incorporated into a fixed inflammatory tissue (oil granuloma) but can still be found in large quantities in the peritoneal fluid for months following its injection. Given that some sort of emulsification and hence solubilization of pristane takes place in vivo, it is clear that cells are exposed to pristane over a prolonged period of time. Although pristane is thought to act indirectly by inducing chronic inflammatory conditions which somehow permit plasmacytomagenesis (11), direct biological effects of the alkane on cells may also contribute to the tumorigenic process. Some direct biological effects of pristane have been described (7).
Pristane (2,6,10,14-tetramethylpentadecane) causes numerous detrimental effects in animals (22, 23, 24, 25). It promotes skin carcinogenesis in C3H mice (26) and the development of 3-methylcholanthrene induced lymphoid malignancies in Copenhagen rats (27). Furthermore, pristane induces histiocytomas in BALB/c and BALB/Mo mice (28) and plasmacytomas in BALB/cAnPt mice (29). Murine plasmacytomagenesis is by far the most widely studied tumor system involving pristane (reviewed in 10, 11). Hence, much of the current interest in this simple compound, an isoprenoid C.sub.19 -isoalkane, is derived from the desire to better understand plasmacytomagenesis in mice.
The tumor inducing activity of pristane, thought to be a non-genotoxic, "complete" carcinogen, is still poorly understood. This is reflected by several diverse hypotheses on its putative effects in vivo. Changes in the conformation of DNA in lymphocytes (30) and hybridoma cells (31) after incorporation of pristane have been observed fluorometrically with propidium iodide. A subsequent fluorescence polarization study revealed pristane-induced changes in the membrane fluidity of rat lymphocytes (32). The generation of oxidation products of pristane by reactive oxygen intermediates (33) had been proposed as a possible additional component of the action of pristane under the conditions of a prolonged inflammation in vivo (34). Immunosuppression (35, 36, 37) and formation of a specific histologic matrix for tumor origin and development (38) after the intraperitoneal administration of pristane may contribute to tumorigenesis through different pathways. Whether or not pristane metabolites that may be generated in vivo (39) are involved in the tumor inducing activity of pristane is presently completely unclear.
In early experiments, attempts were made to solubilize and deliver pristane to cells by using conventional solubilization methods such as mixing with organic solvents and surfactants or by using liposomes of different compositions (13). Both methods were found to be unacceptable. The key constraint derived from the need to deliver sufficient amounts of pristane without exposing the mammalian tester cells to toxic or "bioactive" solvents.
There is no evidence that the complexation of pristane, or any other alkane, with cyclodextrins has ever been used for delivering these compounds in biological assay systems. The predominant examples of the employment of cyclodextrins as solubilizers in biomedicine are in pharmaceutical applications of drug complexation and delivery by cyclodextrins, including non-steroidal anti-inflammatory drugs, steroids, fat-soluble vitamins, prostaglandins, barbiturates, cardiac glycosides, etc. (reviewed in 18). In addition, there are numerous applications of cyclodextrins in the chemical industry such as for separation and resolution of enantiomers as well as for chromatographic separations on cyclodextrin polymers (reviewed in 15 and 16).
Conventional methods of solubilizing alkanes in watery media rely either on the use of organic solvents with or without surfactants, or on use of vesicles such as unilamellar or multilamellar liposomes. The employment of organic solvents imposes severe biological drawbacks because many of them are highly toxic and may produce artifacts even at very low concentrations. Moreover, organic solvents do not guarantee that lipophilic compounds will remain dissolved after further dilution into aqueous media. Although the solubilizing properties of organic solvents may potentially be improved by combining them with surface active compounds (biological detergents, tensids and co-tensids), surfactants frequently alter and eventually disrupt cell membranes. Moreover, it has been shown that the extent of the adsorption of surfactants at oil-water interfaces is largely determined by the nature of the hydrocarbon oil itself (e.g., 87), indicating that the solubilization system must be optimized for each individual alkane under consideration. This would necessitate rather extensive solubilization studies prior to any biological testing. Despite the drawbacks of employing organic solvents and surfactants to introduce alkanes into aqueous media, they are still widely used.
The preparation of lipid vesicles is another widely and successfully used method to deliver highly lipophilic compounds to mammalian cells. Nonetheless, this approach also requires careful consideration of potential artifacts. Problems associated with using liposomes are dependent on the composition of the vesicles. For example, lipid exchange or fusion between vesicles and target cells can alter many properties of the target cell membrane, such as its fluidity or the mobility and activity of membrane bound proteins involved in signal transduction pathways Hence, specific effects of the alkane of interest may be obscured by effects of the liposome itself, thus complicating interpretation of the results. A number of practical disadvantages in employing lipsome vesicles may also be encountered such as problems of reproducibility between different liposome preparations or stability and storage problems. Pristane can be dissolved in unilamellar liposomes, but the effectiveness of the liposomes is dependent upon the phospholipids employed for their preparation. Hence, the solubilization vehicle itself is a variable in the experiments. Thus there is a need to find an inert vehicle to deliver pristane and other alkanes and hydrophobic substances to cells.